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Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1β secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1β. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

Cholinergic control of the heart is exerted by two distinct branches; the autonomic component represented by the parasympathetic nervous system, and the recently described non-neuronal cardiomyocyte cholinergic machinery. Previous evidence has shown that reduced cholinergic function leads to deleterious effects on the myocardium. Yet, whether conditions of increased cholinergic signaling can offset the pathological remodeling induced by sympathetic hyperactivity, and its consequences for these two cholinergic axes are unknown. Here, we investigated two models of sympathetic hyperactivity: i) the chronic beta-adrenergic receptor stimulation evoked by isoproterenol (ISO), and ii) the α2A/α2C-adrenergic receptor knockout (KO) mice that lack pre-synaptic adrenergic receptors. In both models, cholinergic signaling was increased by administration of the cholinesterase inhibitor, pyridostigmine. First, we observed that isoproterenol produces an autonomic imbalance characterized by increased sympathetic and reduced parasympathetic tone. Under this condition transcripts for cholinergic proteins were upregulated in ventricular myocytes, indicating that non-neuronal cholinergic machinery is activated during adrenergic overdrive. Pyridostigmine treatment prevented the effects of ISO on autonomic function and on the ventricular cholinergic machinery, and inhibited cardiac remodeling. α2A/α2C-KO mice presented reduced ventricular contraction when compared to wild-type mice, and this dysfunction was also reversed by cholinesterase inhibition. Thus, the cardiac parasympathetic system and non-neuronal cardiomyocyte cholinergic machinery are modulated in opposite directions under conditions of increased sympathetic drive or ACh availability. Moreover, our data support the idea that pyridostigmine by restoring ACh availability is beneficial in heart disease.